Levoglucosan, cellulosic pyrolysate, is converted to glucose-6-phosphate by a specific levoglucosan kinase in fungi. A novel cDNA of levoglucosan kinase gene (lgk) from yeast Lipomyces starkeyi YZ-215 was isolated by RACE method. The 1,445 bp cDNA fragment (lgk) harbouring the kinase gene exhibited one open reading frame (ORF) composed of 1,317 bp flanked by a 14 bp 5 0 -UTR and a 114 bp 3 0 -UTR, including a 25 bp poly(A) tail. The ORF encoded a 439 amino acid polypeptide with a 48.4 kDa predicted molecular mass. Analysis of amino sequence revealed that the kinase belonged to the bacterial anhydro-N-acetylmuramic acid kinase (AnmK) family, and kinase-like proteins existed in some fungi, especially in filamentous fungi such as Aspergillus. The kinase gene was transformed into Escherichia coli BL21 (DE3), recombinant E. coli could grow in M9 minimal medium with levoglucosan as a sole carbon source when induced by IPTG. In addition, the recombinant kinase was overexpressed, purified and characterized. The kinase was stable at pH 7-10 and showed maximum activity at 30°C and pH 9.0 as natural kinase, but presented higher thermostability. Kinetic constants (apparent K m values) for LG and ATP were 105.3 ± 12.5 and 0.20 ± 0.02 mM, respectively. Furthermore, the kinase showed substrate specificity for LG. This novel levoglucosan kinase gene would be useful in constructing recombinant microbial strains for the efficient bioconversion of cellulosic pyrolysate to ethanol.
Candida antarctica lipase B (CALB) is one of the most widely used and studied enzymes in the world. In order to achieve the high-level expression of CALB in Pichia, we optimized the codons of CALB gene and α-factor by using a de novo design and synthesis strategy. Through comparative analysis of a series of recombinants with different expression components, we found that the methanol-inducible expression recombinant carrying the codon-optimized α-factor and mature CALB gene (pPIC9KαM-CalBM) has the highest lipase production capacity. After fermentation parameters optimization, the lipase activity and protein content of the recombinant pPIC9KαM-CalBM reached 6,100 U/mL and 3.0 g/L, respectively, in a 5-L fermentor. We believe this strategy could be of special interest due to its capacity to improve the expression level of target gene, and the Pichia transformants carrying the codon-optimized gene had great potential for the industrial-scale production of CALB lipase.
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